U.S. patent number 8,198,284 [Application Number 12/410,136] was granted by the patent office on 2012-06-12 for treatment of neurodegenerative disorders with thiourea compounds.
This patent grant is currently assigned to National Health Research Institutes. Invention is credited to Yu-Sheng Chao, Jyh-Haur Chern, Feng-Shiun Shie.
United States Patent |
8,198,284 |
Shie , et al. |
June 12, 2012 |
Treatment of neurodegenerative disorders with thiourea
compounds
Abstract
A method for treating a neurodegenerative disorder. The method
includes administering to a subject in need thereof an effective
amount of one or more thiourea compounds of formula (I) or (II):
##STR00001## Each variable in formula (I) or (II) is defined
herein. Also disclosed is use of these thiourea compounds to reduce
microglia-mediated neuro-inflammation or enhancing microglial
phagocytosis of A.beta..
Inventors: |
Shie; Feng-Shiun (Hsin-Chu,
TW), Chern; Jyh-Haur (Taipei, TW), Chao;
Yu-Sheng (Warren, NJ) |
Assignee: |
National Health Research
Institutes (Miaoli County, TW)
|
Family
ID: |
41257504 |
Appl.
No.: |
12/410,136 |
Filed: |
March 24, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20090275596 A1 |
Nov 5, 2009 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61049052 |
Apr 30, 2008 |
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Current U.S.
Class: |
514/255.03;
514/586; 514/587; 514/514 |
Current CPC
Class: |
A61P
25/28 (20180101); A61K 31/404 (20130101); A61K
31/4965 (20130101); A61P 25/00 (20180101); A61P
25/16 (20180101); A61K 31/17 (20130101) |
Current International
Class: |
A61K
31/17 (20060101); A61K 31/404 (20060101); A61K
31/4965 (20060101); A61P 25/00 (20060101); A61P
25/28 (20060101); A61P 25/16 (20060101) |
Field of
Search: |
;514/255.03,587,586,415 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2056968 |
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Mar 1981 |
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GB |
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2005330284 |
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Feb 2005 |
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JP |
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2005/144790 |
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May 2005 |
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JP |
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WO 99/40088 |
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Aug 1999 |
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WO |
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WO2004/046095 |
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Jun 2004 |
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WO |
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WO2004/096210 |
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Nov 2004 |
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WO |
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WO2005/095345 |
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Oct 2005 |
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WO |
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WO2006/122011 |
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Nov 2006 |
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WO |
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Other References
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examiner .
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2007, 321, 823-29). cited by examiner .
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Primary Examiner: Hui; San-Ming
Assistant Examiner: Cruz; Kathrien
Attorney, Agent or Firm: Occhiuti Rohlicek & Tsao
LLP
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application
Ser. No. 61/049,052 filed Apr. 30, 2008, the content of which is
hereby incorporated by reference in its entirety.
Claims
What is claimed is:
1. A method for treating Alzheimer's disease, comprising
administering to a subject in need thereof an effective amount of
1-naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea.
2. A method for treating Parkinson's disease, comprising
administering to a subject in need thereof an effective amount of
1-naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea.
Description
BACKGROUND
Neurodegenerative diseases afflict more than 30 million of
individuals worldwide. Current therapies target the symptoms of
these diseases, generally with only modest efficacy.
Alzheimer's disease (AD), the most prevalent neurodegenerative
disease, is characterized clinically by progressive memory loss and
cognitive dysfunction, and pathologically by the development in the
brain of intracellular neurofibrillary tangles containing
abnormally hyperphosphorylated tau and extracellular senile amyloid
plaques constituted predominantly of .beta.-amyloid (A.beta.).
Recently, microglia, the resident immune cells of brain, have been
considered to play an important role in the pathogenesis of
neurodegenerative diseases, more specifically, microglia-mediated
neuro-inflammation where microglia are activated has been
implicated in the development of these diseases especially in AD
and Parkinson's disease (PD). However, microglial activation is
associated with production of an assortment of effector molecules
that may have complex and mixed effects on A.beta. clearance and
neuronal survival. Also, based on recent studies, complete
inhibition of microglial activation using non-steroidal
anti-inflammatory drugs appears to show limited therapeutic
benefits for the diseases. See e.g., Shie et al., Current Medicinal
Chemistry, 2007 (14): 2865-2871; Hayden, et. al., Neurology, 2007
(69):275-282; Britschgi et al., Nat. Med., 2007 (13):408-4099; Shie
et al., Brain Pathol., 2005 (15):134-138; Jin et al., Journal of
Neuroinflammation, 2007 (4):2-11; and Gao et al., FASEB J., 2003
(17): 1957-1959.
Fine-tuning microglial activation may confer better means for the
therapy. There is growing consensus that a favorable combination of
reduced microglia-mediated neuro-inflammation and enhanced
phagocytic activity of microglia is essential in slowing the
progression of the neurodegenerative diseases.
SUMMARY
The present invention is based on an unexpected discovery that
certain thiourea compounds are effective in both reducing
microglia-mediated neuro-inflammation and enhancing phagocytosis of
A.beta., which allows these compounds to be applied in treating
neurodegenerative diseases, e.g., AD.
In one aspect, this invention features treating a neurodegenerative
disorder by administering to a subject in need of the treatment an
effective amount of a thiourea compound of formula (I):
##STR00002##
In this formula, A.sub.1 is arylene or heteroarylene; A.sub.2 is
aryl, heteroaryl, H, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, or C(O)R.sub.a,
in which R.sub.a is alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, or
heteroaryl; each of R.sub.1, R.sub.2, and R.sub.3, independently,
is H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, or
C(O)R.sub.b, in which R.sub.b is alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,
aryl, or heteroaryl; or R.sub.1 and R.sub.2, together with the
nitrogen atom to which they are bonded, are heterocycloalkyl; or
R.sub.2 and R.sub.3, together with the two nitrogen atoms to which
they are bonded and the carbon atom bonded to both of the two
nitrogen atoms, are heterocycloalkyl; or R.sub.3, the nitrogen atom
to which it is bonded, and the ring atom of A.sub.1 to which the
nitrogen atom is bonded, together with another ring atom of
A.sub.1, are heterocycloalkyl or heterocycloalkenyl that is fused
with A.sub.1; each of R.sub.4, R.sub.5, R.sub.6, and R.sub.7,
independently, is H, halo, nitro, cyano, amino, hydroxy, alkoxy,
aryloxy, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocycloalkyl, aryl, or heteroaryl; each of X, Y, and Z,
independently, is O, S, SO, SO.sub.2, N(R.sub.c), C(O), C(O)O,
C(O)NR.sub.c, NR.sub.cC(O)NR.sub.d, NR.sub.cC(S)NR.sub.d,
NR.sub.cC(O)O, SO.sub.2NR.sub.c, alkylene, alkenylene, alkynylene,
cycloalkylene, cycloalkenylene, heterocycloalkylene,
heterocycloalkenylene, arylene, or heteroarylene, in which each of
R.sub.c and R.sub.d, independently, is H, alkyl, cycloalkyl,
heterocycloalkyl, aryl, or heteroaryl; each of m and n,
independently, is 0, 1, 2, 3, 4, 5, 6, or 7; and each of x, y, and
z, independently, is 0 or 1.
In particular, this invention features a method for treating
Alzheimer's disease or Parkinson's disease, by administering to a
subject in need thereof an effective amount of a compound of
formula (I) shown above. Referring to formula (I), a subset of the
just-described compounds are those in which X is O and x is 1. In
these compounds, A.sub.1 can be 1,3-phenylene or 1,4-phenylene; Z
can be NHSO.sub.2, NHC(O), C(O)NH, NHC(O)O, NHC(O)NH, NHC(S)NH,
NHC(.dbd.NH)NH, cycloalkylene, or heterocycloalkylene (e.g.,
##STR00003## in which W is O or S and p is 1, 2, or 3); z can be 1;
y can be 0; A.sub.2 can be phenyl, pyridyl, or naphthyl, each of
which is optionally substituted with halo, alkoxy, aryloxy, alkyl,
cycloalkyl, aryl, or heteroaryl; each of R.sub.1, R.sub.2, and
R.sub.3 can be H; each of R.sub.4, R.sub.5, R.sub.6, and R.sub.7
can be H; and each of m and n, independently, can be 0, 1, 2, 3, 4,
or 5; or Z can be NHC(S)NH and A.sub.2 can be naphthyl optionally
substituted with halo, alkoxy, aryloxy, alkyl, cycloalkyl, aryl, or
heteroaryl.
Another subset of the compounds of formula (I) includes those in
which R.sub.3 and the nitrogen atom to which it is bonded, together
with A.sub.1, are
##STR00004## In these compounds, R.sub.1 can be H and R.sub.2 can
be alkyl or C(O)R.sub.b; X can be O and x can be 1; Z can be O, z
can be 0 or 1, and y can be 0; A.sub.2 can be aryl or heteroaryl;
each of R.sub.4, R.sub.5, R.sub.6, and R.sub.7 can be H; and each
of m and n, independently, can be 0, 1, 2, 3, 4, or 5; or R.sub.2
can be alkyl and each of x, y, z can be 0.
Still another subset of the compounds of formula (I) includes those
in which Z is O and z is 1. In these compounds, A.sub.2 can be aryl
(e.g., phenyl optionally substituted with arylamino, halo, alkoxy,
aryloxy, alkyl, cycloalkyl, aryl, or heteroaryl) or heteroaryl;
each of R.sub.1, R.sub.2, and R.sub.3 can be H; or each of R.sub.4,
R.sub.5, R.sub.6, and R.sub.7 can be H and each of m and n,
independently, can be 0, 1, 2, 3, 4, or 5.
The term "treating" or "treatment" refers to administering one or
more thiourea compounds to a subject, who has a neurodegenerative
disorder, a symptom of or a predisposition toward such a disorder,
with the purpose to confer a therapeutic effect, e.g., to cure,
relieve, alter, affect, ameliorate, or prevent the disorder, the
symptom of or the predisposition toward it. Such a subject can be
identified by a health care professional based on results from any
suitable diagnostic method. "An effective amount" refers to the
amount of one or more active thiourea compounds that is required to
confer a therapeutic effect on a treated subject.
In another aspect, this invention features treating a
neurodegenerative disorder by administering to a subject in need of
the treatment an effective amount of a thiourea compound of formula
(II):
##STR00005## In this formula, X is O, N(R.sub.a),
C(R.sub.aR.sub.b), or C(O); in which each of R.sub.a and R.sub.b,
independently, is H, alkyl, cycloalkyl, heterocycloalkyl, aryl, or
heteroaryl; each of R.sub.1, R.sub.2, and R.sub.3, independently,
is H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; or
R.sub.2 and R.sub.3, together with the two nitrogen atoms to which
they are bonded and the carbon atom bonded to both of the two
nitrogen atoms, are heterocycloalkyl; and each of R.sub.4, R.sub.5,
R.sub.6, R.sub.7, R.sub.8, R.sub.9, and R.sub.10, independently, is
H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, halo,
N(R.sub.cR.sub.d), N(R.sub.c)--C(S)--N(R.sub.dR.sub.e);
N(R.sub.c)--C(O)R.sub.d, or N(R.sub.c)--C(O)O--R.sub.d; in which
each of R.sub.c, R.sub.d, and R.sub.e, independently, is H, alkyl,
cycloalkyl, heterocycloalkyl, aryl, or heteroaryl; provided that if
R.sub.10 is at the 3-position, then
##STR00006## is at the 4-position; and if R.sub.10 is at the
4-position, then
##STR00007## is at the 3-position.
In particular, this invention features a method for treating AD or
PD by administering to a subject in need thereof an effective
amount of a compound of formula (II) shown above. For example, one
can administer to a subject having a neurodegenerative disorder a
thiourea compound of formula (II), in which the compound has the
following formula:
##STR00008## In this formula, X, R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, and R.sub.9 are as
defined immediately above.
A subject in need of treatment of a neurodegenerative disorder can
also be concurrently administered with a thiourea compound of the
above formulae and one or more other therapeutic agents. Examples
of such therapeutic agents may include tacrine, donepezil,
galantamine, and rivastigmine, and memantine. The term
"concurrently administered" refers to administering a thiourea
compound and one or more other therapeutic agents at the same time
or at different times during a treatment period.
In still another aspect, this invention features a method of
reducing microglia-mediated neuro-inflammation and/or enhancing
microglial phagocytosis of A.beta. by administering to a subject in
need an effective amount of one or more thiourea compounds of the
above formula (I) or (II). A subject in need can be a patient
having a neurodegenerative disorder, e.g., AD or PD.
The term "alkyl" refers to a straight or branched monovalent
hydrocarbon containing 1-20 carbon atoms (e.g., C.sub.1-C.sub.10).
Examples of alkyl include, but are not limited to, methyl, ethyl,
n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl. The term
"alkylene" refers to a straight or branched bivalent hydrocarbon,
containing 1-20 carbon atoms (e.g., C.sub.1-C.sub.10). Examples of
alkylene include, but are not limited to, methylene and ethylene.
The terms "alkenyl" and "alkenylene" respectively refer to a
straight or branched monovalent and bivalent hydrocarbon containing
2-20 carbon atoms (e.g., C.sub.2-C.sub.10) and one or more double
bonds. Examples of alkenyl and alkenylene include, but are not
limited to, ethenyl, propenyl, propenylene, allyl, and
1,4-butadienyl. The terms "alkynyl" and "alkynylene" respectively
refer to a straight or branched monovalent and bivalent hydrocarbon
containing 2-20 carbon atoms (e.g., C.sub.2-C.sub.10) and one or
more triple bonds. Examples of alkynyl and alkynylene include, but
are not limited to, ethynyl, 1-propynyl, 1- and 2-butynyl, and
1-methyl-2-butynyl. The term "alkoxy" refers to an --O-alkyl
radical. Examples of alkoxy include, but are not limited to,
methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, iso-butoxy,
sec-butoxy, and tert-butoxy.
The terms "cycloalkyl" and "cycloalkylene" respectively refer to a
monovalent and a bivalent saturated hydrocarbon ring system having
3 to 30 carbon atoms (e.g., C.sub.3-C.sub.12). Examples of
cycloalkyl include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylene, cycloheptyl,
and cyclooctyl. The terms "cycloalkenyl" and "cycloalkenylene"
respectively refer to a monovalent and a bivalent non-aromatic
hydrocarbon ring system having 3 to 30 carbons (e.g.,
C.sub.3-C.sub.12) and one or more double bonds. Examples include
cyclopentenyl, cyclohexenyl, and cycloheptenyl. The terms
"heterocycloalkyl" and "heterocycloalkylene" respectively refer to
a monovalent and a bivalent nonaromatic 5-8 membered monocyclic,
8-12 membered bicyclic, or 11-14 membered tricyclic ring system
having one or more heteroatoms (such as O, N, S, or Se). Examples
of heterocycloalkyl and heterocycloalkylene groups include, but are
not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl,
and tetrahydrofuranyl. The term "heterocycloalkenyl" refers to a
monovalent nonaromatic 5-8 membered monocyclic, 8-12 membered
bicyclic, or 11-14 membered tricyclic ring system having one or
more heteroatoms (such as O, N, S, or Se) and one or more double
bonds. The term "heterocycloalkenylene" refers to a bivalent
nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or
11-14 membered tricyclic ring system having one or more heteroatoms
(such as O, N, S, or Se) and one or more double bonds.
The term "aryl" refers to a monovalent 6-carbon monocyclic,
10-carbon bicyclic, 14-carbon tricyclic aromatic ring system.
Examples of aryl groups include, but are not limited to, phenyl,
naphthyl, and anthracenyl. The term "arylene" refers to a bivalent
6-carbon monocyclic, 10-carbon bicyclic, 14-carbon tricyclic
aromatic ring system. The term "aryloxyl" refers to an --O-aryl.
The term "arylamino" refers to an --N(R)-aryl in which R can be H,
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl. The term
"heteroaryl" refers to a monvalent aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having one or more heteroatoms (such as O, N, S, or
Se). Examples of heteroaryl groups include pyridyl, furyl,
imidazolyl, benzimidazolyl, pyrimidinyl, thienyl, quinolinyl,
indolyl, and thiazolyl. The term "heteroarylene" refers to a
bivalent aromatic 5-8 membered monocyclic, 8-12 membered bicyclic,
or 11-14 membered tricyclic ring system having one or more
heteroatoms (such as O, N, S, or Se).
Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl,
cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, alkylene,
alkenylene, alkynylene, cycloalkylene, heterocycloalkylene,
cycloalkenylene, heterocycloalkenylene, arylene, and heteroarylene
mentioned above include both substituted and unsubstituted
moieties. Possible substituents on cycloalkyl, cycloalkenyl,
heterocycloalkyl, heterocycloalkenyl, cycloalkylene,
heterocycloalkylene, cycloalkenylene, heterocycloalkenylene, aryl,
and heteroaryl include, but are not limited to, C.sub.1-C.sub.10
alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl,
C.sub.3-C.sub.20 cycloalkyl, C.sub.3-C.sub.20 cycloalkenyl,
C.sub.1-C.sub.20 heterocycloalkyl, C.sub.1-C.sub.20
heterocycloalkenyl, C.sub.1-C.sub.10 alkoxy, aryl, aryloxy,
heteroaryl, heteroaryloxy, amino, C.sub.1-C.sub.10 alkylamino,
C.sub.1-C.sub.20 dialkylamino, arylamino, diarylamino, hydroxy,
halo, oxo (O.dbd.), thioxo (S.dbd.), thio, C.sub.1-C.sub.10
alkylthio, arylthio, C.sub.1-C.sub.10 alkylsulfonyl, arylsulfonyl,
acylamino, aminoacyl, aminothioacyl, amidino, mercapto, amido,
thioureido, thiocyanato, sulfonamido, guanidine, ureido, cyano,
nitro, acyl, thioacyl, acyloxy, carbamido, carbamyl, carboxyl, and
carboxylic ester. On the other hand, possible substituents on
alkyl, alkenyl, alkynyl, alkylene, alkenylene, or alkynylene
include all of the above-recited substituents except
C.sub.1-C.sub.10 alkyl. Cycloalkyl, cycloalkenyl, heterocycloalkyl,
heterocycloalkenyl, aryl, and heteroaryl can also be fused with
each other.
Shown below are exemplary thiourea compounds that can be used to
practice the method of the invention:
##STR00009## Other thiourea compounds disclosed in U.S. patent
application Ser. Nos. 11/839,326 and 11/839,346, both filed on Aug.
15, 2007, U.S. Provisional Applications 60/910,892 filed on Apr.
11, 2007, 60/942,808 filed on Jun. 8, 2007, and 61/019,663 filed on
Jan. 8, 2008, can also be used to practice the method of this
invention. Methods of synthesizing various thiourea compounds were
also disclosed in the above patent applications.
The thiourea compounds described above include the compounds
themselves, as well as their salts, their solvates, and their
prodrugs, if applicable. A salt, for example, can be formed between
an anion and a positively charged group (e.g., amino) on a thiourea
compound. Suitable anions include chloride, bromide, iodide,
sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate,
methanesulfonate, trifluoroacetate, glutamate, glucuronate,
glutarate, malate, maleate, succinate, fumarate, tartrate,
tosylate, salicylate, lactate, naphthalenesulfonate, and acetate.
Likewise, a salt can also be formed between a cation and a
negatively charged group (e.g., carboxylate) on a thiourea
compound. Suitable cations include sodium ion, potassium ion,
magnesium ion, calcium ion, and an ammonium cation such as
tetramethylammonium ion. The thiourea compounds also include those
salts containing quaternary nitrogen atoms. Examples of prodrugs
include esters and other pharmaceutically acceptable derivatives,
which, upon administration to a subject, are capable of providing
active thiourea compounds.
Also within the scope of this invention is a pharmaceutical
composition containing one or more of the above-described thiourea
compounds for use in treating neurodegenerative disorders, as well
as this therapeutic use and use of the compounds for the
manufacture of a medicament for treating neurodegenerative
disorders.
The details of one or more embodiments of the invention are set
forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and from the claims
DETAILED DESCRIPTION
This invention relates to use of one or more thiourea compounds
described in the summary section above for treating a
neurodegenerative disorder.
The thiourea compounds can be prepared by conventional chemical
transformations (including protecting group methodologies), e.g.,
those described in R. Larock, Comprehensive Organic
Transformations, VCH Publishers (1989); T. W. Greene and P. G. M.
Wuts, Protective Groups in Organic Synthesis, 3.sup.rd Ed., John
Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.
Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995) and subsequent editions thereof. Schemes 1-7
below show transformations for synthesizing certain thiourea
compounds of formula (I) or (II). A.sub.1 and A.sub.2 are defined
above.
The route shown in Scheme 1 exemplifies synthesis of the thiourea
compounds of formula (I) in which Z is NHC(O)NH or NH(CS)NH. O and
S are both denoted as W in this scheme. Dibromo alkyl i is reacted
with a nitro- and hydroxy-substituted aryl or heteroaryl (e.g.,
3-nitrophenol) in the presence of potassium carbonate in
N-methylpyrrolidone (NMP) to form an alkoxy-containing compound ii,
which is subsequently treated with sodium azide to afford azide
compound iii. Reduction of the azide compound leads to amine
compound iv, which is then coupled with aryl isocyanate (or
isothiocyanate) to form a urea (or thiourea) intermediate v.
Subsequent reduction of the nitro group produces amine compound vi,
which is then reacted with thiocarbonyl diimidazole (TCDI),
followed by treatment with 25% aqueous ammonia solution, to afford
thiourea compound vii (e.g., compound 1).
##STR00010##
The route shown in Scheme 2 exemplifies synthesis of the thiourea
compounds of formula (I) in which Z is --NHSO.sub.2--, --NHC(O)--,
or --NHC(O)O--. Amine compound iv is coupled with acyl chloride
(sulfonyl chloride, or chloroformate) to provide compound viii.
Reduction of compound viii with SnCl.sub.2 produces amine compound
ix, which is subsequently reacted with TCDI to afford thiourea
compound x.
##STR00011##
The route shown in Scheme 3 exemplifies synthesis of the thiourea
compounds of formula (I) in which Z is piperazinyl. Coupling bromo
compound ii with piperazine derivative affords compound xi, which
is subsequently converted to amine xii by reducing its nitro group.
Amine xii compound is then reacted with TCDI to afford thiourea
compound x (e.g., compound 2).
##STR00012##
The route shown in Scheme 4 exemplifies synthesis of the thiourea
compounds of formula (I) in which Z is O. Specifically,
3-nitrophenol can first react with a brominated aromatic compound
via a substitution reaction to form an alkoxy-containing compound.
The alkoxy-containing compound can then be reduced (e.g., by
hydrogen or tin chloride) to convert the nitro group to an amino
group. The compound thus formed can then be treated with TCDI and a
base (e.g., ammonia) to form a thiourea compound for practicing the
invention (e.g., compound 3).
##STR00013##
The routes shown in Scheme 5 exemplify synthesis of the thiourea
compounds of formula (I) in which R.sub.3 and the nitrogen atom to
which it is bonded, together with A.sub.1, are a N-containing
bicyclic moiety. Specifically, The N-containing bicyclic moiety can
be prepared by cyclization or by reduction of an aromatic bicyclic
ring. The thiourea moiety of the thiourea compounds can be prepared
by reacting the N-containing bicyclic moiety with TCDI followed by
treatment of amine or ammonium, or by reacting the N-containing
bucyclic moiety with an isothiocyanate (--NCS) compound form the
thiourea compounds for practicing the invention (e.g., compounds
4-6).
##STR00014##
The route shown in Scheme 6 exemplifies synthesis of certain
thiourea compounds of formula (II). Specifically, certain thiourea
compounds can be prepared from a monoamino aromatic compound. For
example, as shown in Scheme 6 below, a monoamino aromatic compound
can react with thiocarbonyl diimidazole, followed by ammonia or a
primary amine, to form a thiourea compound of formula (II).
##STR00015## The route shown in Scheme 7 also exemplifies synthesis
of certain thiourea compounds of formula (II). Specifically,
certain other thiourea compounds can be prepared from a diamino
aromatic compound. For example, as shown in Scheme 7 below, one
amino group on 9H-fluorene-2,7-diamine can first be protected with
a tert-butyloxycarbonyl (Boc) protecting group. The other amino
group 9H-fluorene-2,7-diamine can then react with a halo-containing
compound to form either a compound containing a secondary amino
group or a compound containing a tertiary amino group. The compound
thus formed can be deprotected (e.g., by reacting with
trifluoroacetic acid) and then treated with thiocarbonyl
diimidazole and a base to form a thiourea compound of formula
(II).
##STR00016##
More methods for obtaining various thiourea compounds can be found
in U.S. patent application Ser. Nos. 11/839,326 and 11/839,346, and
U.S. Provisional Applications 60/910,892, 60/942,808, and
61/019,663, supra.
A thiourea compound thus synthesized can be further purified by
flash column chromatography, high performance liquid
chromatography, crystallization, or any other suitable methods.
The thiourea compounds mentioned herein may contain a non-aromatic
double bond and one or more asymmetric centers. Thus, they can
occur as racemates and racemic mixtures, single enantiomers,
individual diastereomers, diastereomeric mixtures, and cis- or
trans-isomeric forms. All such isomeric forms are contemplated.
Also within the scope of this invention is a pharmaceutical
composition contains an effective amount of at least one thiourea
compound described above and a pharmaceutical acceptable carrier.
This invention also covers a method of administering an effective
amount of one or more thiourea compounds to reduce
microglia-mediated neuro-inflammation and/or enhancing phagocytosis
of A.beta. and to treat neurodegenerative disorders such as AD and
PD. Effective doses will vary, as recognized by those skilled in
the art, depending on the route of administration, excipient usage,
and the possibility of co-usage with other therapeutic
treatment.
To practice the method of the present invention, a composition
having one or more thiourea compounds can be administered
parenterally, orally, nasally, rectally, topically, buccally,
vaginally, or via an implanted reservoir. The term "parenteral" as
used herein refers to subcutaneous, intracutaneous, intravenous,
intramuscular, intraarticular, intraarterial, intrasynovial,
intrasternal, intrathecal, intralesional, or intracranial
injection, as well as any suitable infusion technique.
A sterile injectable composition, e.g., a sterile injectable
aqueous or oleaginous suspension, can be formulated according to
techniques known in the art using suitable dispersing or wetting
agents (such as Tween 80) and suspending agents. The sterile
injectable preparation can also be a sterile injectable solution or
suspension in a non-toxic parenterally acceptable diluent or
solvent, for example, as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that can be employed are mannitol,
water, Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium (e.g., synthetic mono- or
diglycerides). Fatty acids, such as oleic acid and its glyceride
derivatives are useful in the preparation of injectables, as are
natural pharmaceutically-acceptable oils, such as olive oil or
castor oil, especially in their polyoxyethylated versions. These
oil solutions or suspensions can also contain a long-chain alcohol
diluent or dispersant, or carboxymethyl cellulose or similar
dispersing agents. Other commonly used surfactants such as Tweens
or Spans or other similar emulsifying agents or bioavailability
enhancers which are commonly used in the manufacture of
pharmaceutically acceptable solid, liquid, or other dosage forms
can also be used for the purposes of formulation.
A composition for oral administration can be any orally acceptable
dosage form including, but not limited to, capsules, tablets,
emulsions and aqueous suspensions, dispersions and solutions. In
the case of tablets for oral use, carriers which are commonly used
include lactose and corn starch. Lubricating agents, such as
magnesium stearate, are also typically added. For oral
administration in a capsule form, useful diluents include lactose
and dried corn starch. When aqueous suspensions or emulsions are
administered orally, the active ingredient can be suspended or
dissolved in an oily phase combined with emulsifying or suspending
agents. If desired, certain sweetening, flavoring, or coloring
agents can be added. A nasal aerosol or inhalation composition can
be prepared according to techniques well known in the art of
pharmaceutical formulation. For example, such a composition can be
prepared as a solution in saline, employing benzyl alcohol or other
suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons, and/or other solubilizing or
dispersing agents known in the art. A thiourea compound-containing
composition can also be administered in the form of suppositories
for rectal administration.
The carrier in the pharmaceutical composition must be "acceptable"
in the sense of being compatible with the active ingredient of the
formulation (and preferably, capable of stabilizing it) and not
deleterious to the subject to be treated. For example, one or more
solubilizing agents, which form more soluble complexes with the
thiourea compounds, can be utilized as pharmaceutical carriers for
delivery of the active thiourea compounds. Examples of other
carriers include colloidal silicon dioxide, magnesium stearate,
sodium lauryl sulfate, and D&C Yellow #10.
Suitable in vitro assays can be used to preliminarily evaluate the
efficacy of the thiourea compounds in reducing microglia-mediated
neuro-inflammation and enhancing phagocytosis of A.beta.. See
Examples 7 and 8 below. The compounds can further be examined for
their efficacy in treating a neurodegenerative disorder. For
example, a compound can be tested in an AD or PD animal model
(e.g., APP transgenic mice or MPTP-treated mice) and clinical
trials. Its therapeutic effects are then assessed. Based on the
results, an appropriate dosage range and administration route can
also be determined.
Without further elaboration, it is believed that the above
description has adequately enabled the present invention. The
following examples are, therefore, to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. All of the publications and patent
applications cited herein are hereby incorporated by reference in
their entirety.
EXAMPLE 1
Synthesis of
1-naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea
(compound 1)
To a stirred solution of 3-nitrophenol (4.17 g, 30.0 mmol) and
1,5-dibromo-pentane (7.59 g, 33.0 mmol) in N-methylpyrolidinone
(100 mL) was added potassium carbonate (6.21 g, 45.0 mmol), and the
resulting mixture was stirred at 90.degree. C. for 6 hours. The
reaction mixture was quenched with water (30 mL) followed by
extraction with ethyl acetate (30 mL.times.3). The combined organic
layers were washed with brine and then concentrated under vacuum.
The residue was purified by silica gel column chromatography to
give 1-(5-bromo-pentyloxy)-3-nitro-benzene (5.10 g, 17.7 mmol, 59%)
as a yellow liquid.
The resulting yellow liquid (3.60 g, 12.5 mmol) was dissolved in
DMSO (20 mL). Sodium azide (1.22 g, 18.7 mmol) was slowly added.
The reaction mixture was stirred overnight at room temperature and
then quenched with water (30 mL) followed by extraction with ether
(30 mL.times.3). The combined organic layers were washed with brine
and then concentrated under vacuum. The residue was purified by
silica gel column chromatography to give
1-(5-azido-pentyloxy)-3-nitro-benzene (3.12 g, 12.5 mmol, 99%) as a
yellow liquid.
To a solution of 1-(5-azido-pentyloxy)-3-nitro-benzene (3.12 g,
12.5 mmol) in 50 mL THF and 1 mL H.sub.2O was added
triphenylphosphine (3.27 g, 12.5 mmol). The reaction mixture was
stirred at room temperature for 48 hours and then was partitioned
with ethyl acetate and water. The aqueous solution was extracted
with ethyl acetate (3.times.50 mL). The combined organic phases
were washed with brine, dried over MgSO.sub.4, and concentrated to
give a yellow liquid, which was purified by silica gel column
chromatography to give 5-(3-nitro-phenoxy)-pentylamine (2.75 g,
12.3 mmol, 98%) as a yellow liquid.
To a solution of 5-(3-nitro-phenoxy)-pentylamine (867 mg, 3.87
mmol) in dichloromethane (5 mL) was added 1-naphthyl isothiocyanate
(788 mg, 4.26 mmol) at room temperature. The reaction mixture was
stirred at room temperature overnight. After removal of the
solvent, the residue was purified by silica gel column
chromatography to give
1-naphthalen-1-yl-3-[5-(3-nitro-phenoxy)-pentyl]-thiourea (1.3 g,
3.18 mmol, 82%) as a yellow gel.
The yellow gel (1.3 g, 3.18 mmol) was dissolved in 30 mL ethanol.
To this solution was added Tin (II) chloride dihydrate (4.05 g,
15.9 mmol). The reaction mixture was stirred at 70.degree. C. for 6
hours. Upon cooling, saturated aqueous sodium bicarbonate solution
was added to adjust the pH value to 7. The solution was then
extracted with ethyl acetate (3.times.50 mL). The combined organic
phases were washed with brine, dried over MgSO.sub.4, and
concentrated to give a yellow gel, which was purified by silica gel
column chromatography eluting with ethyl acetate and n-hexane to
give 1-[5-(3-amino-phenoxy)-pentyl]-3-naphthalen-1-yl-thiourea (1.2
g, 3.17 mmol, 99%) as a light yellow solid.
1-[5-(3-amino-phenoxy)-pentyl]-3-naphthalen-1-yl-thiourea (1.76 g,
4.65 mmol) was mixed with thiocarbonyl diimidazole (993 mg, 5.58
mmol) in dichloromethane (25 mL) and was stirred at room
temperature for 2 hours. 25% aqueous ammonia solution (3 mL) was
added and the reaction mixture was stirred at room temperature
overnight. After removal of the solvent, the residue was purified
by silica gel column chromatography eluting with ethyl acetate and
n-hexane to give Compound 1 (1.7 g, 3.88 mmol, 83%) as a white
solid. MS (EI): m/z 439 (M+H).
EXAMPLE 2
Synthesis of
(3-{5-[4-(4-fluoro-phenyl)-piperazin-1-yl]-pentyloxy}-phenyl)-thiourea
(compound 2)
To a stirred solution of 1-(5-bromo-pentyloxy)-3-nitro-benzene (432
mg, 1.5 mmol) and 1-(4-fluoro-phenyl)-piperazine (297 mg, 1.65
mmol) in acetonitrile (5 mL) was added potassium carbonate (414 mg,
3.0 mmol). After refluxed for 6 hours, the reaction mixture was
quenched with water (10 mL) followed by extraction with ethyl
acetate (10 mL.times.3). The combined organic layers were washed
with brine and then concentrated under vacuum. The resulting
residue was purified by silica gel column chromatography to give
1-(4-fluoro-phenyl)-4-[5-(3-nitro-phenoxy)-pentyl]-piperazine (549
mg, 1.42 mmol, 94%) as a yellow solid.
The obtained compound (549 mg, 1.42 mmol) was dissolved in 5 mL
ethanol. Tin (II) chloride dihydrate (1.81 g, 7.08 mmol) was then
added. The reaction mixture was stirred at 70.degree. C. for 3
hours. Upon cooling, saturated aqueous sodium bicarbonate solution
was added to adjust the mixture's pH value to 7. The solution was
extracted with ethyl acetate (3.times.10 mL), and the combined
organic phases were washed with brine, dried over MgSO.sub.4, and
concentrated to give a white solid, which was purified by silica
gel column chromatography eluting with ethyl acetate-n-hexane to
give
3-{5-[4-(4-fluoro-phenyl)-piperazin-1-yl]-pentyloxy}-phenylamine
(500 mg, 1.40 mmol, 99%) as a white solid.
A solution of
3-{5-[4-(4-fluoro-phenyl)-piperazin-1-yl]-pentyloxy}-phenylamine
(500 mg, 1.40 mmol) and thiocarbonyl diimidazole (299 mg, 1.68
mmol) in dichloromethane (4 mL) was stirred at room temperature for
2 hours. 25% aqueous ammonia solution (2 mL) was added. The
reaction mixture was stirred at room temperature overnight and then
the solvent was removed. The residue was purified by silica gel
column chromatography eluting with ethyl acetate and n-hexane to
give
(3-{5-[4-(4-fluoro-phenyl)-piperazin-1-yl]-pentyloxy}-phenyl)-thiourea
(Compound 19) (425 mg, 1.02 mmol, 73%) as a white solid. MS (EI):
m/z 417 (M+H).
EXAMPLE 3
Synthesis of
{3-[5-(3-Phenylamino-phenoxy)-pentyloxy]-phenyl}-thiourea (compound
3)
Compound 3 was prepared in a manner similar to that outlined in
Scheme 4.
EI-MS (M+1): 422.
EXAMPLES 4-6
Synthesis of 2,3-Dihydro-indole-1-carbothioic acid pentylamide
(compound 4),
N-{4-[5-(4-chloro-phenoxy)-pentyloxy]-2,3-dihydro-indole-1-carbothioy-
l}-4-cyano-benzamide (compound 5), and
N-(5-benzyloxy-2,3-dihydro-indole-1-carbothioyl)-benzamide
(compound 6)
Compound 4-6 were prepared in a manner similar to that outlined in
Scheme 5.
EI-MS (M+1): 249 (compound 4), 520 (compound 5), 389 (compound
6).
EXAMPLE 7
Enhancement of Microglial A.beta. Clearance and Phagocytosis In
Vitro
Compounds were evaluated for their efficacy in enhancing microglial
phagocytosis of A.beta. (or A.beta. clearance). Microglia were
pretreated with a test compound at concentration of 1 .mu.M for 1
hr followed by 24 hr treatment of FITC-labeled A.beta.1-42 (100
nM). Lipopolysaccharide (LPS) at 10 ng/mL was added as a positive
control, which is known to increase A.beta. uptake. The culture
medium was subjected to Western blotting for evaluating residual
A.beta. left in the medium. The mean values of fluorescent
intensity measured by flow cytometer were compared between cells
pretreated with the test compound and the ones in non-treated
controls.
Twenty thiourea compounds, including Compounds 1-6, were tested.
Unexpectedly, treatment with Compounds 1-6 at 1 .mu.M for 24 hr
lowered A.beta. level to approximately 45%-90% of that in
non-treated control; Compound 1 at 10, 20, and 50 .mu.M increased
A.beta. uptake by approximately 29% (n=6, p<0.001), 44% (n=6,
p<0.001), and 128% (n=6, p<0.001), respectively, as compared
to the controls.
EXAMPLE 8
Anti-Inflammatory Activity
Compounds were evaluated for their efficacy in suppressing
microglial immune activation by measuring their potency against
LPS-induced expression of iNOS and COX-2 and IL-6 secretion.
Microglia and Raw 264.7 cells (a microglia-like cell line) were
pretreated with a test compound at various doses for 1 hr followed
by 24 hr treatment of LPS (10 ng/ml). Cell lysates were subjected
to Western blotting for iNOS and COX-2 evaluation. IL-6 secretion
was measured from resulting culture medium by ELISA.
Statistical analysis showed cells pretreated with compound 1 showed
a significant suppression in LPS-induced iNOS and COX-2 expression
in a dose dependent manner, while the house keeping protein,
.beta.-actin, remained unchanged. In addition, compound 1 inhibited
LPS-induced IL-6 secretion by microglia in a dose dependent manner,
which is beneficial for retaining hippocampal functions during
neuro-inflammation. Effective doses of compound 1 to show at least
50% suppression (i.e., IC.sub.50) for iNOS, COX-2, and IL-6 are 1
.mu.M, 50 .mu.M and, 20 .mu.M, respectively.
EXAMPLE 9
Neuro-Protection Against LPS Toxicity
Compounds were evaluated for their efficacy in suppressing
microglia-mediated neurotoxicity. Hippocampal organotypic cultures
were established to serve as an ex vivo model for evaluation of
hippocampal functions. Pre-treatment of the cultures with a test
compound was followed by addition of LPS (10 ng/mL) for 24 hr.
Hippocampal tissues were harvested and subjected to Western
blotting for measuring synaptophysin and post synaptic density
protein (PSD)95 levels, the indications for neuronal synatic
functions.
Unexpectedly, pre-treatment with compound 1 prevented LPS-induced
reduction of synaptophysin and PSD95 levels in hippocampal
organotypic cultures, which indicated that compound 1 may be
neuroprotective against LPS toxicity by suppressing microglial
activation ex vivo.
OTHER EMBODIMENTS
All of the features disclosed in this specification may be combined
in any combination. Each feature disclosed in this specification
may be replaced by an alternative feature serving the same,
equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
From the above description, one skilled in the art can easily
ascertain the essential characteristics of the present invention,
and without departing from the spirit and scope thereof, can make
various changes and modifications of the invention to adapt it to
various usages and conditions. Thus, other embodiments are also
within the scope of the following claims.
* * * * *